1. Field of the Invention
This invention relates to a plasmid containing a gene for tetracycline resistance. More particularly, the present invention relates to a plasmid comprising a gene for tetracycline resistance and a gene for replication which are derived from a glutamic acid-producing coryneform bacterium. The present invention also relates to a DNA fragment containing a gene for tetracycline resistance and a microorganism containing the DNA fragment.
In the present specification, restriction endonucleases (hereinafter often referred to simply as "restriction enzyme") represented by the following abbreviations are restriction endonucleases respectively obtained from the following microorganisms.
______________________________________ Abbreviation Microorganism ______________________________________ EcoRI Escherichia coli RY13 HindIII Haemophilus influenza Rd PstI Providencia stuartii 164 BamHI Bacillus amyloliquefaciens H XbaI Xanthomonas badrii BglII Bacillus globigii SalI Streptomyces albus G HaeIII Haemophilus aegyptius ______________________________________
2. Discussion of Related Art
Conventionally, it is known that a glutamic acid-producing coryneform bacterium produces L-glutamic acid in high yield and that a certain mutant or variant of the glutamic acid-producing coryneform bacterium produces other amino acids such as L-lysine, and purine nucleotides such as an inosinic acid. Such a glutamic acid-producing coryneform bacterium and a mutant or variant thereof have been industrially utilized for producing the amino acids and purine nucleotides as mentioned above.
To obtain the coryneform bacterium in a form capable of producing the desired products efficiently in high yield, breeding of the coryneform bacterium is usually effected. Particularly in recent years, the breeding of the glutamic acid-producing coryneform bacterium by transforming the bacterium with a vector containing a gene coding for a certain peptide desired for the production of the intended amino acid has been attempted using recombinant DNA techniques. To effectively attain the transformation of the glutamic acid-producing coryneform bacterium by means of recombinant DNA techniques, it is necessary to use a vector suitable for the transformation. Therefore, various studies have been made on vectors such as a plasmid and a phage. As the plasmid, there are known those which are obtained from glutamic acid-producing coryneform bacteria, such as a plasmid pCG1 (Japanese Patent Application Laid-open Specification No. 57-134500), a plasmid pCG2 (Japanese Patent Application Laid-open Specification No. 58-35197), a plasmid pCG4 (Japanese Patent Application Laid-open Specification No. 57-183799), plasmids pAM330 and pAM286 (Japanese Patent Application Laid-open Specification No. 58-67699) and a plasmid pHM1519 (Japanese Patent Application Laid-open Specification No. 58-77895). However, most of the above-mentioned plasmids do not have a gene which is useful as a selective marker. In effecting genetic recombination, in order to select a bacterium transformed with a recombinant vector, it is necessary that the recombinant vector has a gene useful as a selective marker, such as a gene for drug resistance. For this reason, the above-mentioned plasmids are lacking in practical utility. In order to overcome the disadvantage as mentioned above, various plasmids having a gene for a drug resistance as a selective marker have heretofore been proposed. For example, as the plasmid having a gene for a drug resistance and derived from a glutamic acid-producing coryneform bacterium, the following plasmids have been proposed.
(1) Plasmid pCG11: The plasmid has as a selective marker a gene for streptomycin and spectinomycin resistances which gene is derived from a plasmid pCG4 of a bacterium belonging to Corynebacterium (Japanese Patent Application Laid-open Specification Nos. 57-183799 and 58-105999).
(2) Plasmids pCB101 and pEthr1: The plasmids have as a selective marker a gene for streptomycin and spectinomycin resistances which gene is derived from the above-mentioned plasmid pCG4 (Japanese Patent Application Laid-open Specification No. 58-105999). As mentioned above, all the plasmids pCG11, pCB101 and pEthr1 commonly have a gene derived from a glutamic acid-producing coryneform bacterium as the selective marker, namely a gene for streptomycin and spectinomycin resistances. In other words, with respect to a selective marker derived from a glutamic acid-producing coryneform bacterium, only the gene for streptomycin and spectinomycin resistances is known.
In general, as mentioned above, in order to surely select a bacterium which has been transformed with a recombinant plasmid, a selective marker is needed. On the other hand, to easily ascertain that the recombinant plasmid which is contained in the transformed bacterium has a gene coding for a certain peptide desired for the production of the intended amino acid (desired peptide), it is necessary that the plasmid contains another selective marker. If a plasmid containing such two kinds of selective markers, namely a gene for a first drug resistance and a gene for a second drug resistance, is used, an intended transformant can be easily selected and separated. Illustratively stated, such a plasmid having the two kinds of selective markers is treated with a specific restriction enzyme so that the plasmid is cleaved at an intermediate position of the gene for a first drug resistance. The cleaved plasmid thus obtained is mixed with a gene for the desired peptide and subjected to incubation. During the incubation, some of the cleaved plasmids are caused to have the gene for the desired peptide inserted between both ends of the cleavage of the plasmid, and some of the cleaved plasmids are caused to be religated at both ends of the cleavage of the plasmid. In the former instance, the gene for a first drug resistance is inactivated (inactivation by insertion), so that when the recombinant plasmid having the gene for the desired peptide is used to transform a bacterium, the obtained transformed bacterium which contains the recombinant plasmid having the gene coding for the desired peptide is no longer resistant to the first drug, that is, sensitive to the first drug but resistant to the second drug. At the latter instance, the resultant plasmid has the uncleaved gene for the first drug resistance and, therefore, when such a plasmid is used to transform a bacterium, the obtained transformed bacterium is resistant to both the first and second drugs. In this connection, it is noted that after the transforming operation, the resultant includes, in addition to the transformed bacterium sensitive to the first drug but resistant to the second drug and the transformed bacterium resistant to both the first and second drugs, a bacterium which has failed to be transformed with any of the abovementioned two kinds of plasmids and hence sensitive to both the first and second drugs.
Accordingly, from the above-mentioned three kinds of bacteria in the transformation system, the bacterium transformed with the recombinant plasmid having the gene for the desired peptide can be easily selected and separated by the criterions of the resistance to the first drug and the resistance to the second drug. In this connection, it should be noted that to attain the easy selection and separation of the bacterium transformed with the recombinant plasmid having the gene for the desired peptide, as described above, it is necessary to employ two kinds of selective markers. However, as the selective marker derived from a glutamic acid-producing coryneform bacterium, there is known only one gene, namely, gene for streptomycin and spectinomycin resistances. With such only one selective marker, the easy selection of the transformed bacterium which contains a plasmid having the gene coding for the desired peptide cannot be attained.
Under such a situation, it has been proposed to use, as a second selective marker having a resistance to a drug other than streptomycin and spectinomycin, a gene derived from other bacterium than a glutamic acid-producing coryneform bacterium. As a plasmid containing such a different gene, the following plasmids may be mentioned.
(1) Plasmid pCE54: The plasmid has as a selective marker a gene for tetracycline resistance, a gene for chloramphenicol resistance and a gene for kanamycin resistance which genes are all derived from Escherichia coli (Japanese Patent Application Laid-open Specification Nos. 58-105999 and 58-126789).
(2) Plasmid pCB101: The plasmid has as a selective marker a gene for kanamycin resistance which is contained in a plasmid pUB110 derived from a bacterium belonging to Staphylococcus (Japanese Patent Application Laid-open Specification No. 58-105999).
(3) Plasmid pEthr1: The plasmid has as a selective marker a gene for expression of threonine operon which gene is derived from Escherichia coli. The use of the plasmid is limited to the transformation of a thr.sup.- strain of the coryneform bacteria (Japanese Patent Application Laid-open Specification No. 58-105999).
(4) Plasmid pAJ43: The plasmid has as a selective marker a gene for chloramphenicol resistance which gene is derived from a plasmid pBR325 of Escherichia coli (Japanese Patent Application Laid-open Specification No. 59-120090).
(5) Plasmid pAJ655: The plasmid has as a selective marker a gene for chloramphenicol resistance which gene is derived from a plasmid pBR325 of Escherichia coli (Japanese Patent Application Laid-open Specification Nos. 58-216199 and 59-120090).
(6) Plasmids pAJ440 and pAJ3148: The plasmid has as a selective marker a gene for kanamycin resistance which gene is derived from a plasmid pUB110 of a bacterium belonging to Staphylococcus (Japanese Patent Application Laid-open Specification No. 58-216199).
However, the second markers contained in the above-mentioned plasmids have the following disadvantage. The above-mentioned second sensitive markers are derived from bacteria other than a glutamic acid-producing coryneform bacterium, that is, the markers are heterogeneous to the coryneform bacterium and, therefore, when such a heterogenous marker is used as the second marker for a host-vector system using as the host a glutamic acid-producing coryneform bacterium, the heterogeneous marker does not sufficiently function and the resistance to the drug is not increased to a sufficient degree. For example, when the gene for tetracycline resistance of the above-mentioned plasmid pCE54, which is derived from E. coli, is used as a selective marker for a host-vector system using, as the host, E.coli to which the gene for tetracycline resistance of the plasmid pCE54 is homogenous, the minimum tetracycline concentration of a medium at which the growth of the E.coli is inhibited (a growth-inhibiting minimum tetracycline concentration) is increased to a value as high as 10 .mu.g/ml. However, when the above-mentioned gene for tetracycline resistance which gene is derived from E.coli is used as a selective marker for a host-vector system using as a host a glutamic acid-producing coryneform bacterium to which the above-mentioned gene for tetracycline resistance is heterogenous, the minimum tetracycline concentration at which the growth of the glutamic acid-producing coryneform bacterium is inhibited is only increased from 0.1 .mu.g/ml to 3.2 .mu.g/ml. As a tetracycline concentration as low as 3.2 .mu.g/ml, the selection of the transformed bacterium cannot be sufficiently attained and there is a danger that the selected transformed bacteria is contaminated with an untransformed bacterium. For the abovementioned reasons, the breeding of a glutamic acid-producing coryneform bacterium is conventionally not effectively effected.